Preparation method of escherichia coli extracellular vesicles and application thereof in anti-breast cancer drugs

Abstract

The application discloses a preparation method of E. coli extracellular vesicles and application of the E. coli extracellular vesicles in anti-breast cancer drugs. The preparation method of the E. coli extracellular vesicles comprises the following steps: knocking out a 3-dehydroquininase gene of E. coli to obtain a recombinant bacterium; lysing the recombinant bacterium and performing low-speed centrifugation to obtain a supernatant and a lysate; performing high-speed centrifugation on the lysate to obtain a precipitate, which is the E. coli extracellular vesicles. The E. coli extracellular vesicles prepared by the method can be applied to the preparation of anti-tumor drugs. The 3-dehydroquininase (aroD) gene of E. coli is singly knocked out to construct a recombinant bacterium strain, and OMVs extracted from the strain can directly inhibit the proliferation of HER2-negative Luminal A type breast cancer and triple-negative breast cancer, but the inhibiting effect on the proliferation of other subtypes of breast cancer is poor. The OMVs prepared by the application can provide a new path for the design and screening of anti-tumor drugs, and have a good drug-making prospect.

Description

Technical Field

[0001] This invention relates to an anti-breast cancer drug, and more particularly to a method for preparing Escherichia coli extracellular vesicles and their application in anti-breast cancer drugs. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Early-stage TNBC is mainly treated with surgical resection and chemotherapy. There is no standard treatment for recurrent and refractory TNBC. However, surgical treatment can lead to increased macromolecular peripheral oxidative damage in the early postoperative period, and chemotherapy often causes adverse reactions and TNBC has low sensitivity to chemotherapy. Overall, it does not improve the survival benefit of patients. Therefore, it is urgent to find a safe and effective treatment strategy for TNBC in clinical practice.

[0003] Bacterial outer-membranevesicles (OMVs) have garnered significant attention in preclinical studies as carriers for delivering antitumor drugs to target cells. OMVs are nanoscale protein-liposomes, small, spherical, bilayered vesicles (10-300 nm) released into the extracellular environment by Gram-negative bacteria. They are composed of lipids, proteins, lipopolysaccharides, phospholipids, DNA, RNA, proteins, an inner membrane, periplasm, and other molecules. These vesicles can transport proteins, virulence factors, lipopolysaccharides, DNA, enzymes, and toxins over long distances to their targets. Numerous studies have utilized biotechnology to design OMVs as carriers for delivering antitumor drugs to target cells. For example, using OMVs to encapsulate mesoporous silica nanoparticles containing 5-FU, co-culturing them with oral squamous cell carcinoma cell lines showed a significant inhibition of tumor cell proliferation. Furthermore, studies have found overexpression of human epidermal growth factor receptor (EGFR) in TNBC cells; by modifying OMVs to increase their affinity for EGFR on the TNBC surface, antitumor drugs can be delivered to TNBC cells. However, there are few reports on the direct inhibitory or killing effects of OMVs on tumor cells. Summary of the Invention

[0004] Objective of this invention: The objective of this invention is to provide a method for preparing Escherichia coli extracellular vesicles, solving the problem of how to prepare Escherichia coli extracellular vesicles with antitumor drug efficacy. Another objective of this invention is to propose an application of Escherichia coli extracellular vesicles in the preparation of antitumor drugs, solving the problem of how to prepare antitumor drugs.

[0005] Technical solution: The present invention provides a method for preparing extracellular vesicles of Escherichia coli, comprising the following steps: (1) The 3-dehydroquinolinease gene of Escherichia coli was knocked out to obtain recombinant bacteria; (2) After lysing the recombinant bacterial cells, centrifuge at low speed and collect the supernatant to obtain the lysate; (3) Centrifuge the lysate at high speed and take the precipitate as the extracellular vesicles of Escherichia coli.

[0006] Preferably, in step (1), the targeted knockout method is CRISPR-Cas9.

[0007] Preferably, in CRISPR-Cas9, the sgRNA sequence is: 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (as shown in SEQ ID No. 1);

[0008] Preferably, in step (2), the method for preparing the recombinant bacterial cells is as follows: Inoculate a single colony of the recombinant bacteria into LB liquid medium and culture by shaking until the bacterial culture reaches OD500. 600 Value ≥ 1; Under aseptic conditions, after centrifuging the bacterial solution, discard the supernatant and take the precipitate, which is the recombinant bacterial cell.

[0009] Preferably, the method for lysing the bacterial cells is as follows: The recombinant bacterial cells were resuspended in lysis buffer to obtain a bacterial suspension, and the bacterial suspension was homogenized to obtain a lysed bacterial suspension.

[0010] Preferably, in step (2), the conditions for low-speed centrifugation are 2-6℃, centrifugation at 1000-3000g for 5-30 minutes.

[0011] Preferably, in step (3), the high-speed centrifugation method is as follows: Centrifuge the lysis solution at 2-6℃ and 9000-11000g for 10-30 min, and collect the supernatant to obtain the first supernatant; Centrifuge the first supernatant at 12000-14000 g for 20-40 min, and then filter and sterilize the supernatant to obtain the second supernatant. Centrifuge the second supernatant at 2-6℃ and 150,000-200,000g for 30-90 minutes, discard the supernatant to obtain the first vesicle precipitate; The first vesicle precipitate was resuspended in buffer solution and centrifuged at 150,000-200,000g for 30-90 minutes at 2-6℃. The supernatant was discarded, and the precipitate was resuspended in buffer solution to obtain E. coli extracellular vesicles.

[0012] The second aspect of this invention discloses the application of the Escherichia coli extracellular vesicles prepared by the above-described method in the preparation of antitumor drugs.

[0013] Preferably, the tumor is breast cancer.

[0014] Furthermore, the breast cancer mentioned is either triple-negative breast cancer or Luminal A type breast cancer.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: This invention constructs a recombinant bacterial strain by knocking out the 3-dehydroquinolinease (aroD) gene in *Escherichia coli*. OMVs extracted from this strain can directly inhibit the proliferation of HER2-negative Luminal A breast cancer and triple-negative breast cancer, but their inhibitory effect on other breast cancer subtypes is poor. The OMVs obtained in this invention can provide a new pathway for the design and screening of anti-tumor drugs and have good prospects for drug development. Attached Figure Description

[0016] Figure 1 This is a morphological image of the extracellular vesicles of Escherichia coli prepared in Example 1. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0018] Example 1: A method for preparing extracellular vesicles of Escherichia coli is as follows: (1) Obtain the complete DNA sequence of the *Escherichia coli* 3-dehydroquinolinease (aroD) gene, aroD [Escherichiacoli str. K-12 substr. MG1655] - Gene ID: 946210. Within the coding region of the aroD gene, select a highly specific sequence of approximately 20 bases: 5'-ATGCGTACCGAGCTTATCGG-3' Based on this sequence, an sgRNA was designed, and its sequence is as follows: 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU-3' Design Donor DNA (design repair template), including: Left homologous arm: a sequence approximately 500-1000 bp upstream of the aroD gene; Right homologous arm: a sequence approximately 500-1000 bp downstream of the aroD gene.

[0019] The specific sequence of the Donor DNA is as follows: The Donor DNA sequence is used to guide cells to completely delete or inactivate the aroD gene after it has been cut.

[0020] (2) Constructing gene editing tools Constructing sgRNA expression plasmids: The designed sgRNA sequence was cloned into the expression vector pTargetF (with spectinomycin resistance) to obtain the recombinant plasmid pTargetF-sgRNA. After entering bacteria, this plasmid continuously transcribes sgRNA.

[0021] The Cas9 enzyme protein was expressed using the pCas plasmid (with kanamycin resistance).

[0022] The left homologous arm, the resistance gene (optional), and the right homologous arm were ligated using overlap extension PCR to obtain the Donor DNA sequence. The PCR product sequence is as follows: (3) The gene editing tool from step (2) was transferred into E. coli to construct aroD gene knockout recombinant bacteria. 80 ng / μL of sgRNA expression plasmid, 150 ng / μL of pCas plasmid, and 250 ng / μL of Donor DNA were added to competent E. coli BL21. After incubation on ice for 20 min, heat shock at 42℃ for 60 s, and recovery at 37℃ for 45 min, transformed E. coli were obtained.

[0023] The transformed BL21 bacteria were plated on double-antibiotic plates containing kanamycin and spectinomycin and incubated at 37°C. Single colonies were picked and added to liquid LB medium for shake culture. Colony PCR was performed using primers targeting the inside and flanking of the aroD gene. The PCR products were sequenced, which confirmed that the aroD gene sequence had been accurately deleted or destroyed.

[0024] The primer sequences are as follows: (1) Internal validation primers (to detect whether the aroD gene has been knocked out) Forward primer: 5'-ATGCGTACCGAGCTTATCGG-3' (target site sequence) Reverse primer: 5'-CCGATAAGCTCGGTACGCAT-3' (target site reverse complementary sequence) (2) External verification primers (to detect whether homologous recombination was successful) Left lateral primer (upstream of the left homologous arm): 5'-GATCTGATCGATCGATCGAT-3' Right lateral primer (downstream of the right homologous arm): 5'-CGATCGATCGATCGATCGAT-3' By changing the temperature or adding an inducer (such as IPTG or arabinose), the pCas and pTargetF plasmids were removed from the identified recombinant bacteria to obtain recombinant bacteria with the aroD gene knocked out.

[0025] (4) The recombinant bacteria with the aroD gene knocked out were inoculated into LB liquid medium and shaken at 37°C and 160 rpm for 12 h until the bacterial culture reached OD. 600 The value is 1; after centrifuging the bacterial culture at 6000 rpm for 5 minutes, discard the supernatant, take 20 μL of bacterial cells and spot them onto an empty nematode growth medium (NGM empty plate). Check for contamination after 12 hours. If no contamination is found, the experiment can continue, and the recombinant bacterial cells should be temporarily stored in a refrigerator at 4℃.

[0026] (5) After resuspending the recombinant bacterial cells in lysis buffer, obtain a bacterial suspension. Pipette 3-4 mL of the bacterial suspension into a dedicated disruption tube, add 1 mL of 0.1 mm magnetic beads, and homogenize the bacterial suspension using a biological homogenizer. The parameters of the biological homogenizer are 6 m / s. Repeat the process twice for 30 seconds, and place the sample on ice during the process. Then centrifuge the bacterial suspension at 2000 g for 10 min at 4°C, and collect the supernatant to obtain the lysis buffer. (6) Centrifuge the lysate at 4°C and 10,000g for 20 min, and take the supernatant to obtain the first supernatant; The first supernatant was centrifuged at 13000 g for 30 min, and the supernatant was filtered through a 0.22 μm filter to remove bacteria, thus obtaining the second supernatant. The second supernatant was centrifuged at 170,000g for 60 minutes at 4°C, and the supernatant was discarded to obtain the first vesicle precipitate. The first vesicle precipitate was resuspended in 1×PBS at a ratio of 1g:20mL, centrifuged at 170000g for 60min at 4℃, the supernatant was discarded, and the precipitate was resuspended in 200μL of 1×PBS to obtain Escherichia coli extracellular vesicles.

[0027] Example 2: Everything else is the same as in Example 1, except that: In step (6), the lysis solution is centrifuged at 2°C and 9000g for 30 min, and the supernatant is taken to obtain the first supernatant. The first supernatant was centrifuged at 12000 g for 40 min, and the supernatant was filtered through a 0.22 μm filter to remove bacteria, thus obtaining the second supernatant. The second supernatant was centrifuged at 2°C and 150,000g for 90 minutes, and the supernatant was discarded to obtain the first vesicle precipitate. The first vesicle precipitate was resuspended in 1×PBS at a ratio of 1g:20mL, centrifuged at 150000g for 90min at 2℃, the supernatant was discarded, and the precipitate was resuspended in 200μL of 1×PBS to obtain Escherichia coli extracellular vesicles.

[0028] Example 3: Everything else is the same as in Example 1, except that: In step (6), the lysis solution is centrifuged at 11000g for 10 min at 6°C, and the supernatant is taken to obtain the first supernatant. The first supernatant was centrifuged at 14000 g for 20 min, and the supernatant was filtered through a 0.22 μm filter to remove bacteria, thus obtaining the second supernatant. The second supernatant was centrifuged at 200,000 g for 30 min at 6 °C, and the supernatant was discarded to obtain the first vesicle precipitate. The first vesicle precipitate was resuspended in 1×PBS at a ratio of 1g:20mL, centrifuged at 200000g for 30min at 6℃, the supernatant was discarded, and the precipitate was resuspended in 200μL of 1×PBS to obtain Escherichia coli extracellular vesicles.

[0029] Comparative Example 1: Everything else is the same as in Example 1, except that: In step (4), the recombinant bacteria with the aroD gene knocked out were replaced with wild-type K-12 Escherichia coli. Wild-type K-12 Escherichia coli extracellular vesicles were finally obtained.

[0030] The microstructure of the aroD single-gene knockout mutant OMVs prepared in Example 1 was examined (TEM), and the results are as follows: Figure 1 As shown.

[0031] The protein concentration of bacterial extracellular vesicles (OMVs) obtained in Examples 1-3 and Comparative Example 1 was determined by the BCA method. OMVs were then added to nematode liquid culture medium at the same final protein concentration (specifically 0.135 μg / μL) for nematode lifespan testing, as follows: After synchronizing GC833 nematodes (tumor model), once the nematodes reached the L4 stage in NGM, 10 nematodes per well were selected and placed into a 96-well plate. The liquid system per well consisted of 120 μL of S medium + 30 pL of FUDR (5-fluorouridine-2'-deoxynucleoside). Each group was fed OMVs every other day, and the number of dead nematodes was counted daily. Nematodes were considered dead when they were upright and unresponsive to platinum needle touch. The blank control group received an equal volume of 1×PBS with OMVs. The blank control group consisted of at least 30 nematodes, and all experiments were repeated at least three times. The results are as follows: Table 1. Effects of different OMVs on the lifespan of nematodes in a tumor model.

[0032] As shown in Table 1, aroD single-gene knockout mutant OMVs significantly prolonged the lifespan of the tumor model nematode GC833. Compared with Comparative Example 1, the lifespan of Example 1 group was significantly prolonged by 28% (p<0.05). There was no significant difference between Comparative Example 1 and the blank control group, indicating that wild-type E. coli OMVs do not have a lifespan-extending function. In tumor model nematodes, abnormal proliferation of tumor cells usually accelerates aging and shortens natural lifespan. The significant lifespan extension of the experimental group's tumor model nematodes may be due to the inhibition of tumor progression, which can serve as indirect evidence that aroD single-gene knockout mutant OMVs have anti-tumor activity.

[0033] The OMVs prepared in Examples 1-3 and Comparative Example 1 were used in anti-tumor proliferation experiments on triple-negative breast cancer (MDA-MB-231 cell line), Luminal A type breast cancer (MCF-7 cell line), and HER2-positive breast cancer (BT-474 cell line). The tumor cell proliferation inhibition rate was determined using the CCK-8 assay. The final concentration of OMVs (based on protein concentration) added to cells in each experimental group was 1 μg / μL. 1×PBS was added to cells in the blank control group.

[0034] The formula for calculating the tumor cell proliferation inhibition rate is: Proliferation inhibition rate = (OD value of blank control group - OD value of experimental group) / (OD value of blank control group - background value of blank well) × 100%.

[0035] The results are as follows: Table 2. Inhibitory effects of different OMVs on the proliferation of different types of breast cancer

[0036] As shown in Table 2, wild-type BL21 OMVs in Comparative Example 1 had no significant inhibitory effect on various breast cancer cell types, while aroD single-gene knockout mutant OMVs in Examples 1-3 only had a good inhibitory effect on triple-negative breast cancer and Luminal A type breast cancer, and had no inhibitory effect on HER2-positive breast cancer.

Claims

1. A method for preparing extracellular vesicles of Escherichia coli, characterized in that, Includes the following steps: (1) The 3-dehydroquinolinease gene of Escherichia coli was knocked out to obtain recombinant bacteria; (2) After lysing the recombinant bacterial cells, centrifuge at low speed and collect the supernatant to obtain the lysate; (3) Centrifuge the lysate at high speed and take the precipitate as the extracellular vesicles of Escherichia coli.

2. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, In step (1), the targeted knockout method is CRISPR-Cas9.

3. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 2, characterized in that, In CRISPR-Cas9, the sgRNA sequence is shown in SEQ ID No. 1; the repair template DNA sequence is shown in SEQ ID No.

2.

4. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, In step (2), the method for preparing the recombinant bacterial cells is as follows: Inoculate a single colony of the recombinant bacteria into LB liquid medium and culture by shaking until the bacterial culture reaches OD500. 600 Value ≥ 1; Under aseptic conditions, after centrifuging the bacterial solution, discard the supernatant and take the precipitate, which is the recombinant bacterial cell.

5. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, The method for lysing the bacterial cells is as follows: The recombinant bacterial cells were resuspended in lysis buffer to obtain a bacterial suspension, and the bacterial suspension was homogenized to obtain a lysed bacterial suspension.

6. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, In step (2), the conditions for low-speed centrifugation are 2-6℃, 1000-3000g for 5-30min.

7. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, In step (3), the high-speed centrifugation method is as follows: Centrifuge the lysis solution at 2-6℃ and 9000-11000g for 10-30 min, and collect the supernatant to obtain the first supernatant; Centrifuge the first supernatant at 12000-14000 g for 20-40 min, and then filter and sterilize the supernatant to obtain the second supernatant. Centrifuge the second supernatant at 2-6℃ and 150,000-200,000g for 30-90 minutes, discard the supernatant to obtain the first vesicle precipitate; The first vesicle precipitate was resuspended in buffer solution and centrifuged at 150,000-200,000g for 30-90 minutes at 2-6℃. The supernatant was discarded, and the precipitate was resuspended in buffer solution to obtain E. coli extracellular vesicles.

8. The use of Escherichia coli extracellular vesicles prepared by any one of claims 1-7 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The tumor is breast cancer.

10. The application according to claim 9, characterized in that, The breast cancer mentioned is one of triple-negative breast cancer or Luminal A type breast cancer.

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